A cell culture model for investigation of synapse influenceability: epigenetics, expression and function of gene targets important for synapse formation and preservation in SH-SY5Y neuroblastoma cells differentiated by retinoic acid.

SH-SY5Y neuroblastoma cells are frequently used for different neuronal cell culture models. As there is no "gold-standard", miscellaneous protocols exist to differentiate these cells into a neuronal cell type. Here, the aim was to find a differentiation condition making cells suitable for investigation of influenceability of synapses by environmental conditions in pharmacologic experiments. For this purpose, effects on synapse molecules should be somehow rateable and cells should be usable for functional analysis like calcium imaging. A system like this is desirable for example in basic research concerning schizophrenia, depression, autism or neurodegeneration as synaptic plasticity and neuronal maturation are known to have a significant impact in these diseases. Cells grown on laminin-coated glass cover slips and treated with 50 µM retinoic acid (RA) turned out to show most convincing morphological signs of neuronal differentiation and attached strongly to the ground, thereby also fulfilling preconditions for functional analysis. Systematic characterisation of this differentiation condition in comparison to non-treated controls revealed lower methylation rates and higher expression of most candidate molecules relevant for formation, preservation and function of synapses as well as differential function. In conclusion, this combination of differentiation strategy and markers seems to be a suitable system to estimate synapse modifications in basic research as it could help to identify possible dedifferentiating effects. To our knowledge, differentiation of SH-SY5Y has not been described as systematic before regarding comprehensiveness of the set of investigated synapse molecules and coverage of applied methods spanning from epigenetics to protein function. Furthermore, this is the first time that SH-SY5Y cells were differentiated on glass cover slips to an extent making them suitable for investigation of synapse molecules as part of stable intercellular connections in downstream functional analyses.

Image movement of the Elekta EPID during gantry rotation: Effects on the verification of dose distributions.

PURPOSE: The use of Electronic Portal Imaging Devices (EPIDs) to acquire dosimetric information, especially for 3D-back-projection, has been increasingly extended. For a precise back-projection, the accurate knowledge of the movement characteristics of the EPID during gantry rotation is an essential requirement. METHODS AND MATERIAL: Measurements were conducted with different alignments of steel balls, which were mounted on the treatment table to avoid secondary effects such as the mechanical sag of gantry or jaws. The image movement of the EPID was determined by comparing the predicted projections of the phantoms with the EPID acquired image. Effects on dosimetric verifications were evaluated by γ-evaluation. RESULTS: The measurement results showed that the shift of the EPID image is larger in Y direction than in X direction. A maximum rotation of 0.3° and nodding of 2.4° of the detector was calculated. Changes in SDD were found up to 10mm. The angles of nodding are overall higher at discrete gantry angles in comparison to images detected for continuous rotation. Using these results we were able to correct the EPID images used for verification measurements. γ-evaluation revealed a significantly improved agreement between planned and measured EPID signal values. CONCLUSION: The measurement methods and algorithms introduced in this study are simple and comprehensive. Using these methods and algorithms we were able to quantify the major effects on geometrical and dosimetric characteristics. This allows the correction of EPID signal measurements for these effects related to the gantry angle, leading to an improved γ-evaluation for treatment plans.

Effect of dose reduction on image registration and image quality for cone-beam CT in radiotherapy.

INTRODUCTION: The additional radiation exposure applied to patients undergoing cone-beam computed tomography (CBCT) for image registration in radiation therapy is of great concern. Since a decrease in CBCT dose is linked to a degradation of image quality, the consequences of dose reduction on the registration process have to be investigated. MATERIAL AND METHODS: This paper examines image quality and registration of low-contrast structures on an Elekta XVI for the two treatment areas prostate and chest while gradually decreasing the mAs per frame and the number of projections per CBCT to achieve dose reduction. RESULTS: Ideal results for image quality were obtained for 1.6 mAs/frame and 377 projections in prostate scans and 0.63 mAs/frame and 440 projections in chest images. Lower as well as higher total mAs lead to a decrease in image quality. In spite of poor image quality, registration can be successfully performed even for lowest possible settings. CONCLUSION: The results for registration allow an extensive dose reduction in both treatment areas. Very low mAs, however, do not qualify for clinical use because subjective judgment of the registration process is impossible. Compared to default presets the use of settings for acceptable image quality already permit a decrease in exposure of about 40 % (29.0 to 16.7 mGy) in prostate scans and 60 % (18.3 to 7.7 mGy) in chest scans.

Quasi-continuous-time impurity solver for the dynamical mean-field theory with linear scaling in the inverse temperature.

We present an algorithm for solving the self-consistency equations of the dynamical mean-field theory (DMFT) with high precision and efficiency at low temperatures. In each DMFT iteration, the impurity problem is mapped to an auxiliary Hamiltonian, for which the Green function is computed by combining determinantal quantum Monte Carlo (BSS-QMC) calculations with a multigrid extrapolation procedure. The method is numerically exact, i.e., yields results which are free of significant Trotter errors, but retains the BSS advantage, compared to direct QMC impurity solvers, of linear (instead of cubic) scaling with the inverse temperature. The new algorithm is applied to the half-filled Hubbard model close to the Mott transition; detailed comparisons with exact diagonalization, Hirsch-Fye QMC, and continuous-time QMC are provided.

Néel transition of lattice fermions in a harmonic trap: a real-space dynamic mean-field study.

We study the magnetic ordering transition for a system of harmonically trapped ultracold fermions with repulsive interactions in a cubic optical lattice, within a real-space extension of dynamical mean-field theory. Using a quantum Monte Carlo impurity solver, we establish that antiferromagnetic correlations are signaled, at strong coupling, by an enhanced double occupancy. This signature is directly accessible experimentally and should be observable well above the critical temperature for long-range order. Dimensional aspects appear less relevant than naively expected.

Perinatal maternal application of Lactobacillus rhamnosus GG suppresses allergic airway inflammation in mouse offspring.

BACKGROUND: Clinical studies indicate that maternal exposure to probiotic bacteria may protect from the development of allergic disease later in life. OBJECTIVE: The purpose of this study was to analyse the effects of a perinatal Lactobacillus rhamnosus GG (LGG) supplementation on the development of allergic disorders in offspring. METHODS: Female BALB/c mice received intragastric LGG every other day before conception, during pregnancy and lactation (perinatal supplementation group) or before conception and during pregnancy only (prenatal supplementation group). Cytokine expression of placental tissues was examined. Offspring of LGG-supplemented and sham-exposed mothers were sensitized to Ovalbumin (OVA), followed by aerosol allergen challenges. Development of experimental asthma was assessed by bronchoalveolar lavage analysis, lung histology and lung function measurement. Cytokine production of splenic mononuclear cells was analysed following in vitro stimulation. RESULTS: Intestinal colonization with LGG was observed in mother mice only, but not in the offspring. However, a reduced expression of TNF-alpha, IFN-gamma, IL-5 as well as IL-10 was observed in mice derived from perinatally LGG-supplemented mothers, whereas IL-13 and IL-4 expression remained unchanged. Moreover, in offspring of prenatally or perinatally LGG-supplemented mothers allergic airway and peribronchial inflammation as well as goblet cell hyperplasia were significantly reduced as compared with mice derived from non-supplemented mothers. In contrast, airway hyperresponsiveness to methacholine was not affected. Exposure to LGG during pregnancy only shifted the placental cytokine expression pattern with a markedly increased TNF-alpha level. CONCLUSION: Our data suggest that LGG may exert beneficial effects on the development of experimental allergic asthma, when applied in a very early phase of life. Immunological effects are, at least in parts, mediated via the placenta, probably by induction of pro-inflammatory cell signals.

Prenatal lipopolysaccharide-exposure prevents allergic sensitization and airway inflammation, but not airway responsiveness in a murine model of experimental asthma.

BACKGROUND: Epidemiological evidence underlines the impact of prenatal environmental factors on the development of postnatal allergies. In this regard an inverse correlation between lipopolysaccharide (LPS) exposure and development of childhood allergy has been found. OBJECTIVE: To assess the impact of prenatal LPS exposure on the development of postnatal respiratory allergies in a mouse model of experimental asthma. METHODS: Female BALB/c mice were exposed to LPS before conception and during pregnancy. Several weeks after birth offspring were sensitized to ovalbumin (OVA) followed by aerosol allergen challenges. RESULTS: Prenatal, maternal LPS-exposure enhanced neonatal IFN-gamma, but not IL-4 and IL-2 production. OVA sensitization of prenatally LPS-exposed mice was accompanied by a marked suppression in anti-OVA IgG1 and IgE as well as unchanged IgG2a antibody responses, paralleled by a significant reduction in IL-5 and IL-13 levels following mitogenic stimulation of splenic leucocytes. Assessment of bronchoalveolar lavage fluids following allergen challenges revealed a marked reduction in eosinophils and macrophages in these mice. Surprisingly, development of airway hyper-responsiveness, a hallmark of bronchial asthma, was not affected. CONCLUSION: This study provides first experimental evidence that LPS may already operate in prenatal life in order to modulate the development of allergies in the offspring.

Flagellar mitochondrial association of the male-specific Don Juan protein in Drosophila spermatozoa.

The Drosophila don juan gene encodes a basic protein (Don Juan protein), which is solely expressed postmeiotically during spermiogenesis in elongated spermatids and in mature sperm. Transgenic expression of a GFP-tagged Don Juan protein (DJ-GFP) in the male germ line showed an association of the fusion protein with the sperm tail. Detailed examination of DJ-GFP localization revealed novel insights into its distinct temporal and spatial distribution along the sperm tail during the last phase of spermatid maturation. Co-localization of DJ-GFP with actin-labeled cysts demonstrated its emergence in elongated spermatids during individualization. Additionally, the endogenous Don Juan protein was detected with epitope-specific antibodies in finally elongated nuclei of spermatids. After completion of nuclear shaping Don Juan is no longer detectable in the sperm heads with the onset of individualization. Mislocalization of the DJ-GFP protein in flagella of a mutant with defective mitochondrial differentiation provides evidence of mitochondrial association of the fusion protein with flagellar mitochondrial arrays. Ectopically expressed DJ-GFP in premeiotic germ cells as well as salivary gland cells confirmed the capability of the fusion protein to associate with mitochondria. Therefore we suppose that Don Juan is a nuclear-encoded, germ-cell specifically expressed mitochondrial protein, which might be involved in the final steps of mitochondrial differentiation within the flagellum.

The Drosophila don juan (dj) gene encodes a novel sperm specific protein component characterized by an unusual domain of a repetitive amino acid motif.

We identified and characterized the don juan gene (dj) of Drosophila melanogaster. The don juan gene codes for a sperm specific protein component with an unusual repetitive six amino acid motif (DPCKKK) in the carboxy-terminal part of the protein. The expression of Don Juan is limited to male germ cells where transcription of the dj gene is initiated during meiotic prophase. But Western blot experiments indicate that DJ protein occurs just postmeiotically. Examination of transgenic flies bearing a dj-promoter-lacZ reporter construct revealed lacZ mRNA distribution resembling the expression pattern of the endogenous dj mRNA in the adult testes, whereas beta-galactosidase expression is exclusively present in postmeiotic germ cells. Thus, these observations strongly suggest that dj transcripts are under translational repression until in spermiogenesis. To study the function and subcellular distribution of DJ in spermiogenesis we expressed a chimaeric dj-GFP fusion gene in the male germline exhibiting strong GFP fluorescence in the liver testes, where only elongated spermatids are decorated. With regard to the characteristic expression pattern of DJ protein and its conspicuous repeat units possible functional roles are discussed.

Effect of secobarbital on theophylline clearance.

Increased clearance of theophylline after the administration of secobarbital was observed in a child receiving phenobarbital. Prior to barbiturate treatment, theophylline clearance was 4.78 ml/kg/min. Beginning 10 days after the institution of secobarbital and phenobarbital therapy, a continually increasing amount of theophylline was required to maintain therapeutic serum concentrations. After 29 days of barbiturate administration, theophylline clearance attained a peak value of 16.1 ml/kg/min, an increase of 337% from the prebarbiturate rate. During this time it was necessary to administer theophylline at a dosage four times above that usually recommended. After secobarbital was discontinued, theophylline clearance returned to 4.53 ml/kg/min. The decrease in theophylline clearance occurred while phenobarbital dosage remained stable. It is apparent that changes in secobarbital dosing were subsequently followed by changes in the clearance of theophylline.